This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Agressive cancer progression is promoted by hypoxic microenvironment and inflammation, conditions that synergize to select for altered survival signals in the tumor cells. Hypoxia may induce cell death that leads to inflammation and/or remodeling of the extracellular matrix, specifically through deposition and processing of extracellular matrix polymer components such as hyaluronan (HA) and collagen. We hypothesize that hypoxia, by altering the redox status of the tumor cell, regulates HA and collagen metabolizing enzymes in an interdependent fashion, and that this link between redox status and matrix remodeling ultimately determines aggressive tumor cell phenotype. In this pilot project, we will test our hypothesis with three specific aims. Aim 1: Determine whether genes involved in HA and collagen metabolism are regulated coordinately in response to oxidative stress imposed by hypoxia and inflammation. We will culture human prostate tumor cells in normoxic and hypoxic conditions in the presence and absence of pro-inflammatory cytokines. We will quantify expression of specific HA synthetic and processing enzymes, as well as enzymes required for proline metabolism, an antioxidant and major component of collagen, in cell culture and in our archived HA-rich and HA-deficient mouse prostate tumors. Dr. DOnald Becker will collaborate with us for these studies. Aim 2: Quantify changes in biosynthesis and turnover of HA and collagen in response to hypoxia and inflammation. We will measure HA accumulation, hyaluronidase activity, collagenase activity and collagen production in tumor cells grown under normoxic and hypoxic conditions as above. Hyaluronidase enzyme functions will be characterized in collaboration with Dr. Joseph Barycki. Cellular redox status will be evaluated by two-photon microscopic quantification of NAD+/NADH ratios and reactive oxygen species (ROS) in collaboration with Dr. Richard Hallworth at Creighton University. Aim 3: Evaluate phenotype of tumor cells with altered HA and collagen metabolism in response to hypoxia and inflammation. We will measure cell proliferation, apoptosis, cell adhesion and motility of tumor cells with manipulated gene expression in hypoxic and normoxic conditions. Specific signaling pathways involved in mediating these responses will be investigated beginning with redox-sensitive transcription factors implicated in motility (CTBP and HIF-1alpha). Defining crosstalk between hypoxia,matrix turnover and tumor cell behavior will elucidate mechanisms by which hypoxia promotes fibrotic tissue damage during tumor progression, a significant clinical problem. Implanting altered tumor cells in mouse prostates and examining colocalization of fibrotic/hypoxic regions with HA and collagen deposition, as well as scoring metastatic frequency, will establish potential clinical relevance. Further delineation of hypoxic gene regulation pathways that mediate cell responses to matrix remodeling will fill in missing links in our understanding of tumorigenesis and metastasis, possibly yielding new therapeutics.